Glucose oxidase enzymatically converts glucose to gluconic acid and hydrogen peroxide. The hydrogen peroxide thus formed can be reduced to H.sub.2 O by a peroxidatively active substance in the presence of an indicator system which is oxidized to produce a response, such as a color change. The chromogenic indicator o-tolidine has been used for some time in glucose test systems, but provides results which are subject to reduction of the oxidized indicator by interfering substances, such as ascorbic acid. Further, the safety of o-tolidine has been questioned.
British published Pat. Nos. 1,464,359 and 1,464,360 which disclose the use of 3,3',5,5' tetramethylbenzidine and similar compounds and their use in the detection and determination of hydrogen peroxide or of constituents which react to form peroxides. Concentrations of the benzidine derivatives disclosed therein are inconsistent and are generally not more than about 6 milligrams/milliliter of impregnating solution used.
The use of the benzidine derivatives at the concentrations disclosed in the aforementioned British published specifications results in relatively slow formation of a color or formation of a color that does not remain stable over a period of time. In the latter case, the developed color tends to deteriorate upon standing, making readings susceptible to error and difficult or impossible by untrained personnel.